This project has focused on investigations regarding the invasiveness of both Borrelia burgdorferi and of Borrelia of relapsing fever. There is an intimate and also required association of Borrelia with the mammalian plasminogen activation system (PAS). The interaction of Borrelia with the PAS provides the spirochetes with borrowed proteolytic activity which in turn enhance invasiveness. The interaction goes even further to include the utilization of the cellular aspects of the PAS to also promote invasiveness and a reduction in inflammation. Studies that demonstrate that Borrelia interacts with each of the PAS components were completed, and documentation was provided for changes in gene expression when Borrelia are stimulated by blood. This project, will consider changes that occur in Borrelia gene expression as a result of contact with blood that result in invasion. In the previous period of this project, we obtained compelling evidence that the biogenesis of critical proteins such as OspC and DbpA were regulated during blood induction by an upregulated Lon-1 protease. The first Specific Aim will study a mechanism whereby blood-induced gene products contribute to invasion by B. burgdorferi. Through microarray experiments, blood markedly induced the upregulation of ospC, dbpA, p35, and lon-1 (among 154 other genes). Lon-1 is an ATP-dependent protease that degrades damaged, senescent, or misfolded proteins. In a lon-1 Borrelia knockout, the levels of OspC, DbpA, and P35 protein were reduced following stimulation with blood even though transcription of these proteins remained elevated. A lon-1 knockout showed an impaired infectivity phenotype and was particularly deficient in invading and colonizing the skin. The possibility that the Lon-1 of Borrelia may have both proteolytic and chaperone functions in the biogenesis of proteins required for invasion will be tested by biochemical (using soluble recombinant wild type and mutant Lon-1) and genetic approaches (using lon-1 knockout and lon-1 complemented strains of infectious B. burgdorferi). Mouse experiments are planned to test the hypothesis that Lon-1 is important in the production of proteins that could be considered to be virulence factors. Since OspC has been shown to be essential for infection of mammals, the hypothesis is that interaction of Lon-1 with OspC (and others to be identified in Specific Aim II) leads to changes in invasion. In Specific Aim II, additional Borrelia responses to stimulation with blood and that lead to invasion will be identified. In this part of the project, the genes associated with the wild type lon-1 and lon-1 mutant (as well as complemented mutants) under stimulation with blood will be identified. Identification of the blood induced regulon will be achieved by proteomic and genomic approaches.